1. Field of the Invention
The present invention relates generally to fluorescence microscopy, and more specifically to providing wide field multi-photon excitation in a confocal plane.
2. Discussion of the Background
Optical microscopy has long been used for inspecting objects too small to be seen distinctly by the unaided eye. Optical microscopy involves providing a light beam incident on a specimen and viewing the light from the specimen through a magnifying lens. Fluorescence microscopy is another type of microscopy in which a fluorescent material is used to mark the specimen of interest, which is then illuminated with a wavelength of light that provides a single photon energy level sufficient to excite the fluorescent material to emit emission light. The image of the specimen is detected by collecting the emission light rather than the excitation light. Fluorescence microscopy can be practiced as standard wide-field microscopy or confocal microscopy.
In wide-field fluorescence microscopy, an excitation light source, such as an arc lamp, provides a parallel or quasi-parallel excitation beam that is converged onto a desired focal plane of the specimen. The image at the focal plane results from all of the light encompassed by the point spread characteristic of a specific objective. Because the point spread function does not define a single plane of focus, excitation of the fluorescent material occurs above and below the desired focal plane and volume information of the specimen cannot be discerned. Computational methods commonly called deconvolution microscopy, which utilize a model of the objective's point spread function, can be used to calculate the light of a specific plane in the specimen from a stack of images taken at different planes of focus. This is done by accounting for the influence of light from each slice upon the other slices to approximate a confocal image slice of defined thickness. The performance of wide-field deconvolution confocal fluorescence microscopy can be similar to optical confocal microscopic methods, however in many cases the resultant image is distorted because of the influence of image noise due to poor contrast caused by background emissions or because the point spread function for the objective may deviate from its respective model under actual experimental conditions. Moreover, these problems make 3-D representations of the specimen difficult to construct.
In confocal fluorescence microscopy, a beam of excitation light is focused on a focal point of the specimen. Where the excitation light has a wavelength sufficient to provide single photon excitation of the fluorescent material, excitation occurs in an hourglass beam waist centered at the focal point which approximates the point spread function of the objective. Unlike wide-field fluorescence microscopy, however, confocality can be obtained by using a pinhole aperture for the excitation source and emission image. Since only parallel light rays that originate from the plane of focus can pass through the pinhole, photons that do not have parallel rays (and are out of the plane of focus) are blocked by the pinhole aperture and do not reach the detector. Thus, the pinhole aperture blocks emission light from above and below the focus point thereby providing a clear image undistorted by information above and below the plane of focus. However, because the emission pinhole provides image data only from the point of focus of the laser beam, the excitation laser beam of a confocal system must be raster scanned in the x and y direction upon the sample and the fluorescent emission intensity collected at each x, y position. From this data an image slice of the specimen can be constructed in a computer. By changing the plane of focus, several images can be obtained and the resulting stack of images can be reconstructed in a computer to obtain a three dimensional (3-D) representation of the specimen.
One common problem with both wide-field and confocal fluorescence microscopy is that single photon excitation of the fluorescent material occurs above and below the point of focus where image of data is actually collected. This unnecessary excitation causes “bleaching” of the material above and below a particular focal plane which when subsequently excited as part of a new focal plane will have reduced emission characteristics. Moreover repeated excitation of tissue above and below the focal plane can damage the tissue, which is particularly undesirable for image creation of live specimens.
Recently, multi-photon fluorescence microscopy has emerged as a new optical sectioning technique for reducing the problems of bleaching and tissue damage. This type of microscopy uses a pulsed illumination laser source having a longer wavelength than required for excitation of the fluorescent material. For example, a dye requiring an excitation wavelength of 500 nm will be illuminated by a laser source operating at 1000 nm such that single photon excitation does not occur in the specimen since the dye does not absorb light at 1000 nm. However, use of a pulsed high-power excitation laser provides a sufficiently high photon density at the point of focus for at least two photons to be absorbed (essentially simultaneously) by the fluorescent material. This absorption of two photons of long wavelength provides excitation energy equivalent to the absorption of a single photon of a shorter wavelength and results in excitation confined to the focal point. Thus with multi-photon excitation, fluorescent material surrounding the focal point is not excited thereby eliminating the need for a pinhole aperture and minimizing problems of bleaching and tissue damage that occur from repeated excitation.
FIG. 6 shows a multi-photon scanning microscopy system disclosed in U.S. Pat. No. 5,034,613. As seen in this figure, the scanning microscope 10 includes an objective lens 12 for focusing incident light 14 from a source 16 such as a laser onto an object plane 18. The illumination provided by incident light beam 14 fills a converging cone generally indicated at 24, the cone passing into the specimen to reach the plane of focus at object plane 18 and form focal point 26. The optical path from laser 16 to the object plane 18 includes a dichroic mirror 28 onto which the light from the laser 16 is directed. The mirror 28 deflects this light downwardly to a mirror 30 which in turn directs the light to a pair of scanning mirrors 32 and 34 by way of curved mirrors 36 and 38. The mirrors 32 and 34 are rotatable about mutually perpendicular axes in order to move the incident light 14 along perpendicular X and Y axes on the object plane so that the stationary specimen is scanned by the incident beam. The light from the scanning mirrors passes through eyepiece 40 and is focused through the objective lens 12 to the object plane 18.
Fluorescence produced in the specimen in the object plane 18 travels back through the microscope 10, retracing the optical path of the incident beam 14, and thus passes through objective lens 12 and eyepiece 40, the scanning mirrors 34 and 32 and the curved mirrors 38 and 36, and is reflected by mirror 30 back to the dichroic mirror 28. The light emitted by fluorescent material in the specimen is at a wavelength that is specific to the fluorophore contained in the specimen, and thus is able to pass through the dichroic mirror 28, rather than being reflected back toward the laser 16, and follows the light path indicated generally at 44. The fluorescent light 42 thus passes through a barrier filter 46 and is reflected by flat mirrors 48, 50 and 52 to a suitable detector such as a photomultiplier tube 54. While not necessary for multi-photon microscopy, an adjustable confocal pin hole 56 is provided in the collection optics 44 to minimize background fluorescence excited in the converging and diverging cones above and below the plane of focus.